It is clinically important to measure protein levels in urine and other body fluids such as serum or cerebrospinal fluid as indicators of the status of patients in various disease states. High levels of protein in urine, for example, can be indicative of renal disease.
Several methods for determining the concentration of protein in urine or cerebrospinal fluid are known in the art. These involve the production of turbidity in acid or alkaline environments, the physical development or enhancement of a chromophore involved in dye binding to the protein, and the chemical generation of chromophores by known procedures such as the biuret or Lowry protein methods.
The biuret and Lowry procedures are generally recognized as being accurate, but are time-consuming and laborious. The other methods are more rapid and convenient, but are less accurate and more subject to interference.
A new method for the determination of protein in urine or cerebrospinal fluid has been described by Iwata, et al., Clinical Chemistry, Volume 25, 1317 (1979). In this method, the urine sample is diluted into a mixture of sodium hydroxide and ethylenediaminetetraacetic acid (EDTA). A cationic surfactant, benzyldimethyl{2-[2-(p-1,1,3,3,-tetramethylbutylphenoxy)ethoxy]ethyl} ammonium chloride (benzethonium chloride) is added immediately and mixed. After allowing the mixture to stand for 40-60 min, the absorbance is measured against water at 450 nm (for cerebrospinal fluid) or at 600 nm (for urine). The absorbance produced by the precipitated protein is compared to standards to determine the protein concentration in the body fluid.
Koreeda, et al., disclose a similar method for determining protein in urine, J. Kyorin Med. Soc., Volume 11, 371 (1980). In this procedure, the urine is diluted into a premixed solution containing sodium hydroxide, EDTA, and benzethonium chloride. The absorbance is measured at 660 nm after 20-40 min and is compared to standards to determine the protein concentration in the body fluid.
Although these two methods provide some improvement over the prior art, they still have several disadvantages. Spectrophotometric interference by sample chromophores and chemical interference by hydrophobic sample constituents such as bilirubin can distort the analysis and lead to false positive results. In addition, the reagent mixture described above is unstable and must be prepared daily. Furthermore, samples containing extremely high protein levels give rise to erroneously low assay values. Finally, the 20-60 min incubation times prior to the measurement of the absorbance are considered a disadvantage in the clinical laboratory.
There is a need for an assay for determining protein in urine and cerebrospinal fluid with improved rapidity and accuracy and which is less prone to interference.